Carbohydrates play a number of extremely important roles in the functioning of living organisms. In addition to their metabolic and storage roles, carbohydrates are covalently attached to numerous other molecules such as proteins and lipids. Molecules such as glycoproteins and glycolipids are generally referred to as glycoconjugates. The biological importance of the carbohydrate portion of glycoconjugates can be seen, for example, in the role they play in affect the ability of glycoproteins to perform their biological functions, including such functions as ligand or receptor recognition.
As a consequence of their diverse and important biological functions, aberrations in the synthesis, degradation, or modification of carbohydrates may give rise to several diseases. Similarly many disease may alter the body's physiology so as to give rise to altered carbohydrate metabolism or the improper glycosylation of proteins, lipids and other glycoconjugates in the body.
Many of the biologically active carbohydrates in the body are polysaccharides and oligosaccharides that are produced in a variety of related forms rather than having a single defined structure. These families of related carbohydrates are frequently found to be components of the same glycoprotein. These families of glycoproteins that share the same polypeptide structure, but display variation in the glycosylation pattern have been referred to as glycoforms, Rademacher, et at, Ann. Rev. Biochem., 57:789-838 (1988).
The relative abundance of members of glycoform family members have been shown to vary in accordance with certain disease states. For example, the disfibrinogenemia associated with liver disease has been associated with variations in the glycosylation of fibrinogens, Martinez, J., et al, Blood, 61:1196-202 (1983), and rheumatoid arthritis has been associated with changes in glycosylation of IgG, Parekh et al. Nature, 316:452-457 (1985).
Diseases based on improper metabolism of carbohydrates from glycoconjugates are well known. The general category of diseases is known by a variety of names, including lysosomal storage disorders, heteroglycanoses, inborn errors of complex carbohydrate metabolism, muoopolysacoharidoses and others. Each of these diseases is the result of a genetic inability to produce one or more of the enzymes required for the stepwise degradation of glycoproteins, mucopolysaccharides or glycolipids, or the carbohydrate portion of said glycoconjugates.
When one of these enzymes in the degradation pathway is incorrectly produced or missing completely, the molecule produced in the last working step of the degradation pathway accumulates due to the body's inability to further cleave the molecule. Over time, the compound that cannot be degraded accumulates to such an extent that it impedes normal biological function in a wide variety of cells throughout the body.
The consequences of this type of genetic defect vary among the different enzyme deficiencies, but the symptoms of these diseases may include organomegaly, corneal opacities, skeletal abnormalities and progressive mental retardation.
The diagnosis of these carbohydrate metabolism disorders has historically been difficult because few methods exist for the separation, detection and identification of a wide variety of complex carbohydrates. The two main methods that have been employed are carbohydrate staining techniques and chromatographic separation and detection methods.
The carbohydrates staining techniques, including the Berry Spot Test and the dimethylmethylene blue (DMB) assay rely on a specific reaction between a chemical dye and a specific class of oligosaccharides. The major application of these methods have been with the mucopolysaccharidoses, which are disorders of glycosaminoglycan degradation. These tests have been proposed for large scale screening, but they are limited to the specific disorders for which the chemistry is designed, and the tests have had a problem with a large number of false positive diagnoses. Sewell, et al, Klin Wochenschr, 57:581-585 (1979), or Lurincz, et al, Ann. Clin. Lab. Sci., 12:258-266 (1982).
Chromatographic separation of oligosaccharides from glycoconjugates has also been proposed as a screening technique for these diseases, but there is no one hromatographic technique or set of chromatographic conditions that will facilitate the separation of the range of carbohydrate-based compounds that accumulate in all of these diseases. The techniques that have been developed include thin layer chromatography (TLC), high performance liquid chromatography and gas chromatography. Each of these methods has some utility in the diagnosis of the carbohydrate metabolic diseases, but they have found limited acceptance in clinical laboratories as a result of their limitations and/or complexity.
Thus it is of interest to provide a general technique for the diagnosis of a variety of diseases characterized by altered levels of carbohydrates in which the diagnostic technique does not require an priori detailed knowledge of the structure of the carbohydrates.